Receptor-mediated uptake of an extracellular bcl-xl fusion protein inhibits apoptosis

ABSTRACT

Apoptosis-modifying fusion polypeptides, and the corresponding nucleic acid molecules, are disclosed. Pharmaceutical compositions comprising these polypeptides, and the use of these polypeptides to modify apoptosis, are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. patent application Ser. No.11/692,112, filed Mar. 27, 2007; which is a continuation of U.S. patentapplication Ser. No. 10/792,517, filed Mar. 2, 2004, now abandoned;which is a divisional of U.S. patent application Ser. No. 09/639,245,filed Aug. 15, 2000, issued as U.S. Pat. No. 6,737,511, on May 18, 2004;which claims the benefit of U.S. Provisional Application No. 60/149,220,filed Aug. 16, 1999. All of these listed patent documents areincorporated by reference in their entirety.

FIELD

This invention relates to modification of the apoptotic response oftarget cells, for instance target cells in a subject. More specifically,it relates to apoptosis-modifying fusion proteins with at least twodomains, one of which targets the fusion protein to a target cell, andanother of which modifies an apoptotic response of the target cell.

BACKGROUND

Tissue and cell homeostasis in multicellular organisms is largelyinfluenced by apoptosis, the phenomenon of programmed cell death bywhich an intra- or extra-cellular trigger causes a cell to activate abiochemical “suicide” pathway. Morphological indicia of apoptosisinclude membrane blebbing, chromatin condensation and fragmentation, andformation of apoptotic bodies, all of which take place relatively earlyin the process of programmed cell death. Degradation of genomic DNAduring apoptosis results in formation of characteristic, nucleosomesized DNA fragments; this degradation produces a diagnostic 180 bpladdering pattern when analyzed by gel electrophoresis. A later step inthe apoptotic process is degradation of the plasma membrane, renderingapoptotic cells leaky to various dyes (e.g., trypan blue and propidiumiodide). Apoptotic cells are usually engulfed and destroyed early in thedeath process; thus, apoptosis tends not to be associated withinflammation caused by cytoplasm leakage, as is found in necrosis.

Various in vivo triggers can induce apoptosis; the paradigmatic triggeris a shortage of one or more necessary growth factors. Apoptosis plays asignificant role in development of the neural system (reviewed in Cowanet al., Science 225:1258-1265, 1984; Davies, Development 101:185-208,1987; Oppenheim, Annu. Rev. Neurosci. 14:453-501, 1991) and lymphoidsystem (reviewed in Blackman et al., Science 248:1335-1341, 1990;Rothenberg, Adv. Immunol. 51:85-214, 1992) of vertebrates. Systemdevelopment occurs through selective apoptotic extinction of certaincell populations.

In spite of much study, the molecular mechanisms of apoptosis are notfully elucidated. It does appear, however, that different apoptosisinducers may trigger different apoptotic pathways. For instance, certainpathways are transcription-dependent, in that apoptosis requires thesynthesis of new proteins after stimulation by, for instance, withdrawalof growth factors. Staurosporine, a non-specific kinase inhibitor, incontrast, stimulates a transcription-independent pathway. Transcriptiondependent and independent pathways appear to share downstreamcomponents, including the ICE family of proteases (caspases). See Rubin,British Med. Bulle., 53:617-631, 1997, for a review of apoptosis inneurons; More general reviews include Ashkenazi and Dixit, Science281:1305-1308; Thornberry and Lazebnik, Science 281:1312-1316; and Adamsand Cory, Science 281:1322-1326.

Apoptosis is recognized as a gene-directed event, controlled by acomplex set of interacting gene products that inhibit or enhanceapoptosis (Williams and Smith, Cell 74:777-779, 1993; reviewed in White,Genes Dev. 10:1-15, 1996). Extensive effort is currently underway toidentify and characterize the genes involved in this process. The firstprotein characterized as influencing apoptosis was Bcl-2 (Cleary et al.,Cell 47:19-28, 1986; Tsujimoto and Croce, Proc. Natl. Acad. Sci. USA83:5214-5218, 1986). Since its discovery, several Bcl-2-related proteins(the Bcl-2 family of proteins) have been identified as being involved inregulation of apoptosis (White, Genes Dev. 10:1-15, 1996; Yang et al.,Cell 80:285-291, 1995). One such is Bcl-x, which is expressed in twodifferent forms, long (Bcl-x_(L)) and short (Bcl-x_(S)) (Boise et al.,Cell 74:597-608, 1993).

Bcl-x_(L) and certain other members of the Bcl-2 family are, like Bcl-2itself, powerful inhibitors of cell death (the “anti-death” Bcl-2 familymembers). Genetic overexpression of Bcl-2 has been shown to blockapoptosis in the nervous system of transgenic mice (Chen et al., Nature385:434-439, 1997; Henkart, Immunity 4:195-201, 1996;Lippincott-Schwartz et al., Cell 67:601-616, 1991; Hunziker et al., Cell67:617-627, 1991; Krajewski et al., Cancer Research 53:4701-4714, 1993;Martinou et al., Neuron 13:1017-1030, 1994).

Other members of the Bcl-2 protein family, including Bcl-x_(S), Bad andBax, are potent enhancers of apoptosis and therefore toxic to cells(“pro-death” Bcl-2 family members). Though the mechanism of apoptosisinduction by these proteins remains unknown, it has been suggested thatBad binding to Bcl-x_(L) may promote cell death (Yang et al., Cell80:285-291, 1995; Zha et al., J Biol. Chem 272:24101-24104, 1997) andthat phosphorylation of Bad may prevent its binding to Bcl-x_(L),thereby blocking cell death (Zha et al., J Biol. Chem. 272:24101-24104,1997; Zha et al., Cell 87:619-628, 1996).

In addition to its involvement in neuronal and lymphoid systemdevelopment and overall cell population homeostasis, apoptosis alsoplays a substantial role in cell death that occurs in conjunction withvarious disease and injury conditions. For instance, apoptosis isinvolved in the damage caused by neurodegenerative disorders, includingAlzheimer's disease (Barinaga, Science 281:1303-1304), Huntington'sdisease, and spinal-muscular atrophy. There is also a substantialapoptotic component to the neuronal damage caused during stroke episodes(reviewed in Rubin, British Med. Bulle., 53(3):617-631, 1997; andBarinaga, Science 281:1302-1303), and transient ischemic neuronalinjury, as in spinal cord injury. It would be of great benefit toprevent undesired apoptosis in various disease and injury situations.

Treatment with standard apoptosis inhibitory molecules, for instancepeptide-type caspase inhibitors (e.g., DEVD-type), though useful forlaboratory experiments where microinjection can be employed, has provenunsatisfactory for clinical work due to low membrane permeability ofthese inhibitors. Transfection of cells with various native proteins,including members of the Bcl-2 family of regulatory proteins, has dualdisadvantages. First, transfection is usually not cell-specific, andthus may disrupt apoptotic processes non-specifically in all cells.Second, transfection tends to provide long term alterations in theapoptotic process, in that once a transgene is integrated and functionalin the genome of target cells, it may be difficult to turn off.Especially in instances of stroke episodes or transient ischemicneuronal injury, it would be more advantageous to be able to applyapoptosis regulation for short periods of time. Therefore, there isstill a strong need to develop pharmaceutical agents that overcome thesedisadvantages.

Cancer and other hyper-proliferative cell conditions can be viewed asinappropriate escape from appropriate cell death. As such, it would beadvantageous to be able to enhance apoptosis in certain of these cellsto stop unregulated or undesired growth. Various attempts have been madeto selectively eliminate cancerous cells through the use of targetedimmunotoxins (genetic or biochemical fusions between a toxic molecule,for instance a bacterial toxin, and a targeting domain derived,typically from an antibody molecule).

One bacterial toxin that has been employed in attempts to kill cancerouscells is diphtheria toxin (DT). Diphtheria toxin has three structurallyand functionally distinct domains: (1) a cell surface receptor bindingdomain (DTR), (2) a translocation domain (DTT) that allows passage ofthe active domain across the cell membrane, and (3) the A (enzymaticallyactive) chain that, upon delivery to a cell, ADP-ribosylates elongationfactor 2 and thereby inactivates translation. Altering the receptorspecificity of the diphtheria toxin has been used to generate toxinsthat may selectively kill cancer cells in vitro (Thorpe et al., Nature271:752-755, 1978) and in man (Laske et al., Nature Medicine3:1362-1368, 1997). Promising though they might have seemed, these andsimilar hybrid immunotoxins have proven to be substantially lesseffective at targeted cell death than the toxins from which they weregenerated. This is perhaps due to difficulties in translocation of thefusion protein into the target cell (Columbatti et al., J. Biol. Chem.261:3030-3035, 1986). In addition, in vivo results have beenparticularly poor using such hybrid constructs (Fulton et al., Fed.Proc. 461:1507, 1987).

It is to biological molecules that overcome deficiencies in the priorart that the present invention is directed.

SUMMARY OF THE DISCLOSURE

Disclosed herein are apoptosis-modifying fusion proteins constructed byfusing a protein, or an apoptosis-modifying fragment or variant thereof,from the Bcl-2 protein family with a cell-binding, targeting domain suchas one derived from a bacterial toxin. Using this approach,apoptosis-modifying fusion proteins can be delivered effectivelythroughout the body and targeted to select tissues and cells. In certainembodiments, fusing various cell-binding domains to Bcl-2 familyproteins (such as Bcl-x_(L) or Bad) allows targeting to specific subsetsof cells in vivo, permitting treatment and/or prevention of thecell-death related consequences of various diseases and injuries. Thedelivery of other Bcl-2 homologues to the cell permits regulation ofcell viability either positively (using anti-death Bcl-2 familymembers), or negatively (using pro-death members of the Bcl-2 family).

The apoptosis-modifying fusion proteins disclosed herein havespecifiable cell-targeting and apoptosis-modifying activities. Thus,they may be used clinically to treat various disease and injuryconditions, through inhibition or enhancement of an apoptotic cellularresponse. For instance, apoptosis-inhibiting fusion proteins arebeneficial to minimize or prevent apoptotic damage that can be caused byneurodegenerative disorders (e.g., Alzheimer's disease, Huntington'sdisease, spinal-muscular atrophy), stroke episodes, and transientischemic neuronal injury (e.g., spinal cord injury). Theapoptosis-enhancing fusion proteins n can be used to inhibit cellgrowth, for instance uncontrolled cellular proliferation.

Accordingly, a first embodiment is a functional apoptosis-modifyingfusion protein capable of binding a target cell, having a first domaincapable of modifying apoptosis in the target cell, and a second domaincapable of specifically targeting the fusion protein to the target cell.This fusion protein further integrates into or otherwise crosses acellular membrane of the target cell upon binding to that cell.

Certain embodiments will also include a linker between these twodomains. This linker will usually be at least 5 amino acids long, forexample between 5 and 100 amino acids in length, and may for instanceinclude the amino acid sequence shown in SEQ ID NO: 6. Appropriatelinkers may be 6, 7, or 8 amino acids in length, and so forth, includinglinkers of about 10, 20, 30, 40 or 50 amino acids long.

The apoptosis modifying fusion proteins may also include a third domainfrom one of the two original proteins, or from a third protein. Thisthird domain may improve the fusion protein's ability to be integratedinto or otherwise cross a cellular membrane of the target cell. Anexample of such a third domain is the translocation region (domain orsub-domain) of diphtheria toxin.

Target cells for the fusion proteins disclosed herein include, but arenot limited to, neurons, lymphocytes, stem cells, epithelial cells,cancer cells, neoplasm cells, and others, including otherhyper-proliferative cells. The target cell chosen will depend on whatdisease or injury condition the fusion protein is intended to treat.

Receptor-binding domains may be derived from various cell-type specificbinding proteins, including for instance bacterial toxins (e.g.,diphtheria toxin or anthrax toxin), growth factors (e.g., epidermalgrowth factor), monoclonal antibodies, or single-chain antibodiesderived from antibody genes. Further, variants or fragments of suchproteins may also be used, where these fragments or variants maintainthe ability to target the fusion protein to the appropriate target cell.

Further specific embodiments employ essentially the entire Bcl-x_(L)protein as the apoptosis-modifying domain of the fusion protein, orvariants or fragments thereof that maintain the ability to inhibitapoptosis in a target cell to which the protein is exposed. Examples ofsuch proteins are fusion proteins made of the Bcl-x_(L) protein,functionally linked to the diphtheria toxin receptor binding domainthrough a peptide linker of about six amino acids. One such protein isBcl-x_(L)-DTR, which consists of Bcl-x_(L) and DTR, without thetranslocation domain of diphtheria toxin. The nucleotide sequence ofthis fusion protein is shown in SEQ ID NO: 1, and the correspondingamino acid sequence in SEQ ID NOs: 1 and 2.

Another such example is LF_(n)-Bcl-x_(L), which includes the aminoterminal portion (residues 1-255) of mature anthrax lethal factor (LF),coupled to residues 1-209 of Bcl-x_(L). The nucleotide sequence of thisfusion protein is shown in SEQ ID NO: 7, and the corresponding aminoacid sequence in SEQ ID NOs: 7 and 8.

Also encompassed are fusion proteins wherein the apoptosis-modifyingdomain is an apoptosis-enhancing domain. Such domains include thevarious pro-death members of the Bcl-2 family of proteins, for instanceBad, and variants or fragments thereof that enhance apoptosis in atarget cell. A specific appropriate variant of the Bad protein has anamino acid other than serine at amino acid position 112 and/or position136, to provide constitutively reduced phosphorylation.

Thus, one specific embodiment is a functional apoptosis-enhancing fusionprotein capable of binding a target cell, comprising the Bad protein andthe diphtheria toxin translocation and receptor binding domains,functionally linked to each other. The Bad protein of this embodimentcan also contain a mutation(s) at position 112 and/or 136 to change theserine residue to some other amino acid, to reduce phosphorylation ofthe protein. One such protein is Bad-DTTR; the nucleotide sequence ofthis protein is shown in SEQ ID NO: 3, and the corresponding amino acidsequence in SEQ ID NOs: 3 and 4.

Also disclosed herein are nucleic acid molecules encodingapoptosis-modifying fusion proteins, for instance the nucleic acidsequences in SEQ ID NOs: 1, 3, and 7, and nucleic acid sequences havingat least 90% sequence identity to these sequences, for instance thoseencoding for proteins containing one or more conservative amino acidsubstitutions. Other nucleic acid sequences may have 95% or 98% sequenceidentity with SEQ ID NO: 1, 3, or 7. Also encompassed are recombinantnucleic acid molecules in which such a nucleic acid sequence is operablylinked to a promoter, vectors containing such a molecule, and transgeniccells comprising such a molecule.

Methods also are provided for producing functional recombinantapoptosis-modifying fusion proteins capable of binding to a target cell,integrating into or otherwise translocating across the cell membrane,and modifying an apoptotic response of the target cell. Such a proteincan be produced in a prokaryotic or eukaryotic cell, for instance bytransforming or transfecting such a cell with a recombinant nucleic acidmolecule comprising a sequence which encodes a disclosed bispecificfusion protein. Appropriate eukaryotic cells include yeast, algae, plantor animal cells. Such transformed cells can then be cultured underconditions that cause production of the fusion protein, which is thenrecovered through protein purification means. The protein can include amolecular tag, such as a six histidine (hexa-his) tag, to facilitate itsrecovery.

Protein analogs, derivatives, or mimetics of the disclosed proteins,which retain the ability to target to appropriate target cells andmodify apoptosis in those cells, are also encompassed in embodiments.

Compositions containing these apoptosis modifying fusion proteins, andanalogs, derivatives, or mimetics of these proteins, are further aspectsof this disclosure. Such compositions may further contain apharmaceutically acceptable carrier, various other medical ortherapeutic agents, and/or additional apoptosis modifying substances.

Methods for modifying apoptosis in a target cell are also encompassed,wherein a sufficient amount of a fusion protein of the currentdisclosure to modify apoptosis in the target cell is contacted with atarget cell. Modification of apoptosis can be by either inhibition orenhancement of an apoptotic response of the target cell. The fusionprotein can be administered to the target cell in the form of apharmaceutical composition, and can further be administered with variousmedical or therapeutic agents, and/or additional apoptosis modifyingsubstances. Such agents may include, for instance, chemotherapeutic,anti-inflammatory, anti-viral, and antibiotic agents.

Bcl-x_(L)-DTR, LF_(n)-Bcl-x_(L), or related fusion proteins can be usedto inhibit apoptosis in a target cell by contacting the target cell withan amount of this protein sufficient to inhibit apoptosis.Alternatively, Bad-DTTR or related fusion proteins can be used toenhance apoptosis in a target cell by contacting the target cell with anamount of this protein sufficient to enhance apoptosis.

A specific aspect disclosed herein is the method of reducing apoptosisin a subject after transient ischemic neuronal injury, for instance aspinal cord injury, comprising administering to the subject atherapeutically effective amount of an apoptosis-inhibiting proteinaccording to this disclosure. Examples of such fusion proteins includeBcl-x_(L)-DTR and LF_(n)-Bcl-x_(L). These proteins can be administeredin the form of a pharmaceutical composition, and can be co-administeredwith various medical or therapeutic agents, and/or additional apoptosismodifying substances.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description of severalembodiments, which proceeds with reference to the accompanying figuresand tables.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the construction, production and bioactivity ofBcl-x_(L)-DTR and Bcl-x_(L) transfected into HeLa cells. FIG. 1A is aschematic representation of construction of Bcl-x_(L)-DTR. FIG. 1B is aWestern blot of the lysates of HeLa cells transiently transfected withBcl-x_(L) (lane b) and Bcl-x_(L)-DTR (lane c). Lane a containsuntransfected cells as a control. A small amount of endogeneousBcl-x_(L) is present in lanes a and c. FIG. 1C is a graph that showstransient transfection of Bcl-x_(L) (◯) and Bcl-x_(L)-DTR (⋄) genes intoHeLa cells inhibits apoptotic cell death induced by the addition of STS.Apoptosis in control cells transfected with the vector (pcDNA3) vectoris shown for comparison (□).

FIG. 2 is a graph that shows the results of a diphtheria toxin receptorcompetitive binding assay. Cold competitor proteins [native DT (Δ),Bcl-x_(L)-DTR (▴), Bcl-x_(L) (◯), and DTR ()] were used to displaceI¹²⁵ labeled diphtheria toxin (DT) tracer, and the amount of bound,labeled tracer was measured. Native DT and the fusion proteinBcl-x_(L)-DTR compete for DT receptor binding in the nanomolarconcentration range.

FIG. 3 depicts the results of several experiments that demonstrate theapoptosis-inhibiting character of the fusion construct Bcl-x_(L)-DTR.Panel A is a graph of a time course of apoptosis induced bystaurosporine (STS). Cells were treated with 0.1 μM STS (◯), 0.1 μM STSplus 4.8 μM Bcl-x_(L)-DTR protein medium (Δ), or 20 μl of PBS (□).Results are presented as the average number of apoptotic cells per field(magnification 160×). For each point, at least 5 fields were counted ineach of at least 3 wells. FIG. 3B is a SDS-PAGE gel that shows thatBcl-x_(L)-DTR prevents PARP cleavage. Lane a contains control HeLa cellsnot incubated with STS (uninduced cells); Lane b, HeLa cells treatedwith STS plus 1 μM Bcl-x_(L)-DTR protein; Lane c, HeLa cells treatedwith STS plus 1.48 μM Bcl-x_(L)-DTR protein; and Lane d, HeLa cellstreated with STS and no fusion protein.

FIG. 4 shows that Bcl-x_(L)-DTR inhibits of apoptosis induced byγ-radiation, but not that induced by α-Fas antibody. FIG. 4A is a graphshowing that the addition of Bcl-x_(L)-DTR prior to irradiation ofJurkat cells reduces apoptotic death in response to γ-radiation. Controlcells were not irradiated and not treated with Bcl-x_(L)-DTR. FIG. 4B isa graph that shows that, in Jurkat cells, Bcl-x_(L)-DTR had littleinhibitory effect on apoptosis induced by anti-Fas antibody. Controlcells were treated with PBS and no anti-Fas antibody.

FIG. 5 shows that Bcl-x_(L)-DTR inhibits apoptosis induced bypoliovirus.

FIG. 6 is a graph showing the time course of viability of cells treatedwith Bad-DTTR.

FIG. 7 shows the results of experiments that demonstrate that Bad-DTTRcombined with STS triggers massive cell death. FIG. 7A is a graphquantifying cell death after treatment of U251 MG cells with variouscombinations of STS and Bad-DTTR. Apoptosis is most enhanced when cellsare treated with 0.1 μM STS plus 0.65 μM Bad-DTTR, and cells begin todie about 12 hours after treatment. In the experiment depicted in FIG.7B, the use of 1 μM STS in combination with various concentrations ofBad-DTTR cause an earlier onset of apoptosis in U251 MG cells. Key:□=PBS; ⋄=0.1 μM STS; ◯=0.65 μM Bad-DTTR; Δ=0.065 μM Bad-DTTR;

=0.1 μM STS+0.65 μM Bad-DTTR; ⊖=0.1 μM STS+0.065 μM Bad-DTTR.

FIG. 8 is a schematic diagram of the chimera LF_(n)-Bcl-x_(L). Thefusion gene, LF_(n)-Bcl-x_(L), was inserted into the vector, pET15b,yielding a histidine tag sequence at the N terminus of theLF_(n)-Bcl-x_(L) gene.

FIG. 9 is a graph showing the time course of apoptosis induced by STS inJ774 cells, with or without LF_(n)-Bcl-x_(L) protein. J774 cells at3×10⁴/cm² were treated with 0.1 μM staurosporine alone, 0.1 μMstaurosporine along with LF_(n)-Bcl-x_(L) (28 μg/ml) plus PA (33 μg/ml),or with PBS alone. The apoptotic and living cells were stained withHoechst 33342 and counted at the indicated times, and the data werecalculated as reported (Liu et al., Proc. Natl. Acad. Sci. USA 96:9563-9567, 1999).

FIG. 10 is a bar graph showing the effect of LF_(n)-Bcl-x_(L) againstJ774 treated with STS. J774 cells at 10⁴/cm² were treated with PBS, 0.1μM staurosporine alone, 0.1 μM staurosporine along with LF_(n) (28μg/ml), 0.1 μM staurosporine along with Bcl-x_(L) (28 μg/ml), 0.1 μMstaurosporine along with LF_(n)-Bcl-x_(L) (28 μg/ml), 0.1 μMstaurosporine along with LF_(n)-Bcl-x_(L) (28 μg/ml) plus PA (33 μg/ml),0.1 μM staurosporine along with PA (33 μg/ml) and 0.1 μM staurosporinealong with LF_(n) (28 μg/ml) plus PA (33 μg/ml). The apoptotic andliving cells were stained with Hoechst 33342 48 hours later and counted,and the data were calculated as for FIG. 9.

FIG. 11 is a bar graph showing the effect of LF_(n)-Bcl-x_(L) againstJurkat cells treated with STS. Jurkat cells at 10⁵/ml were treated with0.1 μM staurosporine alone, 0.1 μM staurosporine along withLF_(n)-Bcl-x_(L) (28 μg/ml) plus PA (33 μg/ml) or with PBS. Theapoptotic and living cells were stained with Hoechst 33342 21 hourslater and counted, and the data were calculated as for FIG. 9.

FIG. 12 is a bar graph showing that the fusion protein LF_(n)-Bcl-x_(L)prevents apoptosis by in neonatal rat retinal ganglion cells 24 hoursafter optic nerve section. The apoptotic and living cells in retinalganglion layers were counted 24 hours after optic nerve sectionimmediately followed by the injection of PBS or the indicatedprotein(s). The percentage of apoptotic cells versus total retinalganglion cells per retina is represented.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 shows the DNA coding sequence and corresponding amino acidsequence of Bcl-x_(L)-DTR.

SEQ ID NO: 2 shows the amino acid sequence of Bcl-x_(L)-DTR.

SEQ ID NO: 3 shows the DNA coding sequence and corresponding amino acidsequence of Bad-DTTR.

SEQ ID NO: 4 shows the amino acid sequence of Bad-DTTR.

SEQ ID NO: 5 shows the nucleotide sequence of the linker used to linkBcl-x_(L) to DTR in the fusion construct Bcl-x_(L)-DTR.

SEQ ID NO: 6 shows the amino acid sequence of the linker used to linkBcl-x_(L) to DTR to form Bcl-x_(L)-DTR.

SEQ ID NO: 7 shows the DNA coding sequence and corresponding amino acidsequence of LF_(n)-Bcl-x_(L).

SEQ ID NO: 8 shows the amino acid sequence of LF_(n)-Bcl-x_(L).

DETAILED DESCRIPTION OF THE INVENTION I. Abbreviations and Definitions

A. Abbreviations

DT: diphtheria toxin

DTR: diphtheria toxin receptor binding domain

DTT: diphtheria toxin translocation domain

DTTR: diphtheria toxin translocation and receptor binding domains

E. coli: Escherichia coli

EF: anthrax edema factor

LF: anthrax lethal factor

LF_(n): first 255 residues of anthrax lethal factor

moi: multiplicity of infection

PA: anthrax protective antigen

PCR: polymerase chain reaction

RE: restriction endonuclease

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

STS: staurosporine

TdT: terminal deoxyribonucleotidyl transferase

TUNEL: TdT-dependent dUTP-biotin nick end labeling

B. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes V published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al., (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). The nomenclature for DNAbases and the three-letter code for amino acid residues, as set forth at37 CFR § 1.822, are used herein.

In order to facilitate review of the various embodiments of theinvention, the following definitions of terms are provided. Thesedefinitions are not intended to limit such terms to a scope narrowerthan would be known to a person of ordinary skill in the field.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Apoptosis-modifying ability: A protein has apoptosis-modifying abilityif it is capable of modifying apoptosis in a cell. This ability isusually measurable, either in vivo or in vitro, using any one of myriadapoptosis assays. The art is replete with methods for measuringapoptosis. Appropriate techniques include dye exclusion (e.g. Hoechstdye No. 33342), assaying for caspase activity, and TUNEL-staining. Thespecific ability of a fusion protein to modify the apoptotic response ofa cell to various apoptosis-inducing stimuli can be determined byrunning standard apoptosis assays in the absence of or presence ofvarious concentrations of the fusion proteins. The results of the assayare then compared, and can be reported for instance by presenting thepercentage of apoptosis that occurs in the presence of the fusionprotein.

The invention also includes analogs, derivatives or mimetics of thedisclosed fusion proteins, and which have apoptosis-modifying ability.Such molecules can be screened for apoptosis-modifying ability byassaying a protein similar to the disclosed fusion protein, in that ithas one or more conservative amino acid substitutions or short in-framedeletions or insertions, or analogs, derivatives or mimetics thereof,and determining whether the similar protein, analog, derivative ormimetic provides modification of apoptosis in a desired target cell. Theapoptosis-modifying ability and target cell binding affinity of thesederivative compounds can be measured by any known means, including thosediscussed in this application.

Apoptosis-modifying fusion protein: Proteins that have at least twodomains fused together, at least one domain comprising a cell bindingregion capable of targeting the fusion protein to a target cell (thetargeting or cell-binding domain), and at least one domain capable ofmodifying apoptosis in the target cell (the apoptosis-modifying domain).The apoptosis-modifying fusion proteins of the current invention arefurther characterized by their ability to integrate into or otherwisecross a cellular membrane of the target cell when deliveredextracellularly. An apoptosis-modifying fusion protein is consideredfunctional if it targets to the correct target cell, and modifies anapoptotic response of that cell.

In general, the two domains of the disclosed fusions are geneticallyfused together, in that nucleic acid molecules that encode each proteindomain are functionally linked together, for instance directly orthrough the use of a linker oligonucleotide, thereby producing a singlefusion-encoding nucleic acid molecule. The translated product of such afusion-encoding nucleic acid molecule is the apoptosis-modifying fusionprotein.

Apoptosis-modifying fusion proteins can be labeled according to how theyinfluence apoptosis in the target cell. For instance, anapoptosis-modifying fusion protein according to the current inventionthat inhibits apoptosis in the target cell can be referred to as anapoptosis-inhibiting fusion protein (e.g., Bcl-x_(L)-DTR andLF_(n)-Bcl-XL). Likewise, if the fusion protein enhances apoptosis inthe target cell, it can be referred to as an apoptosis-enhancing fusionprotein (e.g., Bad-DTTR). Specific apoptosis-modifying fusion proteinsare usually named for the proteins from which domains are taken to formthe fusion, or from the domains actually used. For instance,“Bcl-x_(L)-DTR” (SEQ ID NOs: 1 and 2) consists of the entire Bcl-x_(L)protein fused in frame to the receptor-binding domain of diphtheriatoxin (DTR) via a short linker.

A Bcl-2 protein: A Bcl-2 protein is a protein from the Bcl-2 family ofproteins and includes those proteins related to Bcl-2 by sequencehomology, which affect apoptosis. By way of example, the family includesBcl-2, Bcl-x (both the long and short forms), Bax, and Bad. Additionalmembers of the Bcl-2 family of proteins are known (Adams and Cory,Science 281:1322-1326, 1998).

Molecules that are derived from proteins of the Bcl-2 family includefragments of such proteins (e.g., fragments of Bcl-x_(L) or Bad),generated either by chemical (e.g., enzymatic) digestion or geneticengineering means. Such fragments may comprise nearly all of the nativeprotein, with one or a few amino acids being genetically or chemicallyremoved from the amino or carboxy terminal end of the protein, orgenetically removed from an internal region of the sequence.

Derived molecules, or derived from: The term “X-derived molecules” or“derived from X,” where X is a protein also encompasses analogs(non-protein organic molecules), derivatives (chemically functionalizedprotein molecules obtained starting with the disclosed proteinsequences) or mimetics (three-dimensionally similar chemicals) of thenative protein structure, as well as proteins sequence variants orgenetic alleles, that maintain biological functionality. Where thederived molecule is used as the targeting domain of anapoptosis-modifying fusion protein, the biological functionalitymaintained is the ability to target to fusion protein to the desiredtarget cell. Likewise, where the derived molecule is used as theapoptosis-modifying domain of the fusion, the functionality maintainedis the ability to affect apoptosis in the target cell. Each of thesefunctionalities can be measured in various ways, including specificprotein binding and apoptosis assays, respectively.

Injectable composition: A pharmaceutically acceptable fluid compositioncomprising at least one active ingredient, e.g., an apoptosis-modifyingfusion protein. The active ingredient is usually dissolved or suspendedin a physiologically acceptable carrier, and the composition canadditionally comprise minor amounts of one or more non-toxic auxiliarysubstances, such as emulsifying agents, preservatives, and pH bufferingagents and the like. Such injectable compositions that are useful foruse with the fusion proteins of this invention are conventional;appropriate formulations are well known in the art.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Linker: A peptide, usually between two and 150 amino acid residues inlength, which serves to join two protein domains in a multi-domainfusion protein. Peptide linkers are generally encoded for by acorresponding oligonucleotide linker. This can be genetically fused, inframe, between the nucleotides that encode the domains of a fusionprotein.

Oligonucleotide: A linear polynucleotide sequence of between six and 300nucleotide bases in length.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Parenteral: Administered outside of the intestine, e.g., not via thealimentary tract. Generally, parenteral formulations are those that willbe administered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, or subcutaneously,and various surface applications including intranasal, intradermal, andtopical application, for instance.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Martin, Remington'sPharmaceutical Sciences, published by Mack Publishing Co., Easton, Pa.,15th Edition, 1975, describes compositions and formulations suitable forpharmaceutical delivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified fusionprotein preparation is one in which the fusion protein is more enrichedthan the protein is in its generative environment, for instance within acell or in a biochemical reaction chamber. Preferably, a preparation offusion protein is purified such that the fusion protein represents atleast 50% of the total protein content of the preparation. More purifiedpreparations will have fusion protein that represents at least 60%, 70%,80% or 90% of the total protein content.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination can be accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

Similarly, a recombinant protein is one encoded for by a recombinantnucleic acid molecule.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences is expressed in terms of the similarity betweenthe sequences, otherwise referred to as sequence identity. Sequenceidentity is frequently measured in terms of percentage identity (orsimilarity or homology); the higher the percentage, the more similar thetwo sequences are. Homologs of the apoptosis-modifying fusion proteinwill possess a relatively high degree of sequence identity when alignedusing standard methods. For instance, encoding sequences encompassed inthe current invention include those that share about 90% sequenceidentity with SEQ ID NO: 1 and NO: 3.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, PNAS. USA 85:2444, 1988;Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS5:151-153, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huanget al., Comp. Appls Biosci. 8:155-65, 1992; and Pearson et al., Meth.Mol. Biol. 24:307-31, 1994. Altschul et al., Nature Genet. 6:119-29,1994, presents a detailed consideration of sequence alignment methodsand homology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) orLFASTA (Pearson and Lipman, PNAS. USA 85:2444, 1988) may be used toperform sequence comparisons (Internet Program© 1996, W. R. Pearson andthe University of Virginia, “fasta20u63” version 2.0u63, release dateDecember 1996). ALIGN compares entire sequences against one another,while LFASTA compares regions of local similarity. These alignment toolsand their respective tutorials are available on the Internet at the NCSAweb-site.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at least90%, at least 92%, at least 94%, at least 95%, at least 97%, at least98%, or at least 99% sequence identity.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Conditions for nucleic acid hybridization andcalculation of stringencies can be found in Sambrook et al., InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989;and Tijssen, Laboratory Techniques in Biochemistry and Molecular BiologyPart I, Ch. 2, Elsevier, New York, 1993. Nucleic acid molecules thathybridize to the disclosed apoptosis-modifying fusion protein sequencesunder stringent conditions will typically hybridize to a probe (based onthe entire fusion protein encoding sequence, an entire domain, or otherselected portions of the encoding sequence) under wash conditions of0.2×SSC, 0.1% SDS at 65° C.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that encode substantially the same protein.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a Bcl-x_(L)-DTR-specific binding agent bindssubstantially only the Bcl-x_(L)-DTR protein in a specific preparation.As used herein, the term “Bcl-x_(L)-DTR-specific binding agent” includesBcl-x_(L)-DTR antibodies and other agents that bind substantially onlyto a Bcl-x_(L)-DTR protein in that preparation.

Anti-Bcl-x_(L)-DTR antibodies may be produced using standard proceduresdescribed in a number of texts, including Harlow and Lane (UsingAntibodies, A Laboratory Manual, CSHL, New York, 1999, ISBN0-87969-544-7). The determination that a particular agent bindssubstantially only to Bcl-x_(L)-DTR protein may readily be made by usingor adapting routine procedures. One suitable in vitro assay makes use ofthe Western blotting procedure (described in many standard texts,including Harlow and Lane, 1999). Western blotting may be used todetermine that a given protein binding agent, such as ananti-Bcl-x_(L)-DTR monoclonal antibody, binds substantially only to theBcl-x_(L)-DTR protein.

Alternately, because the disclosed apoptosis-modifying proteins arefusion proteins, they can be detected using antibodies to one or theprotein domains used in their construction. For instance, fusionscontaining Bcl-x_(L) can be detected using the monoclonal antibody 2H12(Hsu and Youle, J. Biol. Chem. 272:13829-13834, 1997; now available fromNeo Markers, Union City, Calif., clone #2H121-3) or other professionallyavailable antibody preparations, for instance, polyclonalanti-Bcl-x_(L)/x_(S) #06-851 from Upstate Biotechnology, Lake Placid,N.Y.; polyclonal rabbit anti-Bcl-x_(L) #65189E from PharMingen, SanDiego, Calif.; and rabbit polyclonal (#B22630-050/B22630-150) or mousemonoclonal (B61220-050/B61220-150) anti-Bcl-x_(L) from TransductionLaboratories, Lexington, Ky.). Antibodies that recognize diphtheriatoxin are, for instance, available from the Centers for Disease Control,Atlanta, Ga.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) that bindto Bcl-x_(L)-DTR would be Bcl-x_(L)-DTR-specific binding agents.

Target cell binding affinity: The physical interaction between a targetcell and an apoptosis-modifying fusion protein as disclosed in thisinvention can be examined by various methods. Alternatively, the abilityof fusion protein to compete for binding to its target cell with eithernative targeting domain or antibody that recognizes the targeting domainbinding site on the target cell can be measured. This allows thecalculation of relative binding affinities through standard techniques.

Therapeutically effective amount of an apoptosis-modifying fusionprotein: A quantity of apoptosis-modifying fusion protein sufficient toachieve a desired effect in a subject being treated. For instance, thiscan be the amount necessary to measurably inhibit or enhance apoptosisin a target cell.

An effective amount of apoptosis-modifying fusion protein may beadministered in a single dose, or in several doses, for example daily,during a course of treatment. However, the effective amount of fusionprotein will be dependent on the fusion protein applied, the subjectbeing treated, the severity and type of the affliction, and the mannerof administration of the fusion protein. For example, a therapeuticallyeffective amount of fusion protein can vary from about 0.01 mg/kg bodyweight to about 1 g/kg body weight.

The fusion proteins disclosed in the present invention have equalapplication in medical and veterinary settings. Therefore, the generalterm “subject being treated” is understood to include all animals (e.g.,humans, apes, dogs, cats, horses, and cows), and particularly mammals,that are or may suffer from a chronic or acute condition or injury thatcauses apoptosis, or a lack thereof, susceptible to modification usingmolecules of the current invention.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Transgenic cell: A transgenic cell is one that has been transformed witha recombinant nucleic acid molecule.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

II. Construction, Expression, and Purification of Apoptosis-ModifyingFusion Proteins

A. Selection of Component Domains.

This invention provides generally an apoptosis-modifying fusion proteinthat binds to a target cell, translocates across or otherwise integratesinto the membrane(s) of the target cell, and modifies an apoptoticresponse of the target cell. As such, any target cell in which it isdesirous to modify (either inhibit or enhance) apoptosis is anappropriate target for a bispecific fusion protein. The choice ofappropriate protein binding domain for incorporation into the disclosedapoptosis-modifying fusion protein will be dictated by the target cellor cell population chosen. Examples of targeting domains include, forinstance, nontoxic cell binding domains or components of bacterialtoxins (such as diphtheria toxin or anthrax toxin), growth factors (suchas epidermal growth factor), monoclonal antibodies, cytokines, and soforth, as well as targeting competent variants and fragments thereof.

The choice of appropriate Bcl-2 family member-derivedapoptosis-modifying domain will depend on the manner in which the targetcell's response to apoptosis is to be modified. Where apoptosis is to beinhibited by the resultant fusion protein, anti-death members of theBcl-2 protein family are appropriate sources for apoptosis-modifyingdomains. One such fusion protein is Bcl-x_(L)-DTR, which employs thelong form of Bcl-x, Bcl-x_(L), as the apoptosis-modifying domain.Alternately, where enhancement of apoptosis is desired, pro-deathmembers of the Bcl-2 family of proteins will be appropriate. Forinstance, Bad-DTTR employs the pro-death protein Bad as itsapoptosis-modifying domain.

Translocation of the apoptosis-modifying fusion protein into the targetcell is important. A translocation domain may be included in the fusionprotein as a separate, third domain. This could be supplied from a thirdprotein, unrelated to the cell-binding and apoptosis-modifying domains,or be a translocation domain of one of these proteins (e.g., thediphtheria toxin translocation (DTT) domain used in Bad-DTTR). The DTTdomain contains several hydrophobic and amphipathic alpha helices and,after insertion into cell membranes, creates voltage dependent ionchannels (Kagan et al., Proc Natl Acad Sci USA 78:4950-4954, 1981;Donovan et al., Proc Natl Acad Sci USA 78:172-176, 1981).

Alternately, the translocation function can be provided through the useof a cell-binding domain or apoptosis-modifying domain that confers theadditional functionality of membrane translocation or integration. Thisis true in Bcl-x_(L)-DTR, wherein Bcl-x_(L) provides both theapoptosis-modifying ability and translocation into the cell.

B. Assembly

The construction of fusion proteins from domains of known proteins iswell known. In general, a nucleic acid molecule that encodes the desiredprotein domains are joined using standard genetic engineering techniquesto create a single, operably linked fusion oligonucleotide. Appropriatemolecular biological techniques may be found in Sambrook et al., InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989.Specific examples of genetically engineered multi-domain proteins,including those joined by various linkers, can be found in the followingpatent documents:

U.S. Pat. No. 5,834,209 to Korsmeyer;

U.S. Pat. No. 5,821,082 to Chinnadurai;

U.S. Pat. No. 5,696,237 to FitzGerald et al.;

U.S. Pat. No. 5,668,255 to Murphy;

U.S. Pat. No. 5,587,455 to Berger et al.;

WO 98/17682 to Korsmeyer; and

WO 98/12328 to Home et al.

It will usually be convenient to generate various control molecules forcomparison to an apoptosis-modifying fusion protein, in order to measurethe specificity of the apoptosis modification provided by each fusionprotein. Appropriate control molecules may include one or more of thenative proteins used in construction of the fusion, or fragments ormutants thereof.

C. Expression

One skilled in the art will understand that there are myriad ways toexpress a recombinant protein such that it can subsequently be purified.In general, an expression vector carrying the nucleic acid sequence thatencodes the desired protein will be transformed into a microorganism forexpression. Such microorganisms can be prokaryotic (bacteria) oreukaryotic (e.g., yeast). One appropriate species of bacteria isEscherichia coli (E. coli), which has been used extensively as alaboratory experimental expression system. A eukaryotic expressionsystem will be preferred where the protein of interest requireseukaryote-specific post-translational modifications such asglycosylation. Also, protein can be expressed using a viral (e.g.,vaccinia) based expression system.

Protein can also be expressed in animal cell tissue culture, and such asystem will be appropriate where animal-specific protein modificationsare desirable or required in the recombinant protein.

The expression vector can include a sequence encoding a synthesistargeting peptide, positioned in such a way as to be fused to the codingsequence of the apoptosis-modifying fusion protein. This allows theapoptosis-modifying fusion protein to be targeted to specificsub-cellular or extra-cellular locations. Various appropriateprokaryotic and eukaryotic targeting peptides, and nucleic acidmolecules encoding such, are well known to one of ordinary skill in theart. In a prokaryotic expression system, a signal sequence can be usedto secrete the newly synthesized protein. In a eukaryotic expressionsystem, the targeting peptide would specify targeting of the hybridprotein to one or more specific sub-cellular compartments, or to besecreted from the cell, depending on which peptide is chosen. Throughthe use of a eukaryotic secretion signal sequence, theapoptosis-modifying fusion protein can be expressed in a transgenicanimal (for instance a cow, pig, or sheep) in such a manner that theprotein is secreted into the milk of the animal.

Vectors suitable for stable transformation of culturable cells are alsowell known. Typically, such vectors include a multiple-cloning sitesuitable for inserting a cloned nucleic acid molecule, such that it willbe under the transcriptional control of 5′ and 3′ regulatory sequences.In addition, transformation vectors include one or more selectablemarkers; for bacterial transformation this is often an antibioticresistance gene. Such transformation vectors typically also contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, and atranscription termination site, each functionally arranged in relationto the multiple-cloning site. For production of large amounts ofrecombinant proteins, an inducible promoter is preferred. This permitsselective production of the recombinant protein, and allows both higherlevels of production than constitutive promoters, and enables theproduction of recombinant proteins that may be toxic to the expressingcell if expressed constitutively.

In addition to these general guidelines, protein expression/purificationkits are produced commercially. See, for instance, the QIAexpress™expression system from QIAGEN (Chatsworth, Calif.) and variousexpression systems provided by INVITROGEN (Carlsbad, Calif.). Dependingon the details provided by the manufactures, such kits can be used forproduction and purification of the disclosed apoptosis-modifying fusionproteins.

D. Purification

One skilled in the art will understand that there are myriad ways topurify recombinant polypeptides, and such typical methods of proteinpurification may be used to purify the disclosed apoptosis-modifyingfusion proteins. Such methods include, for instance, proteinchromatographic methods including ion exchange, gel filtration, HPLC,monoclonal antibody affinity chromatography and isolation of insolubleprotein inclusion bodies after over production. In addition,purification affinity-tags, for instance a six-histidine sequence, maybe recombinantly fused to the protein and used to facilitate polypeptidepurification. A specific proteolytic site, for instance athrombin-specific digestion site, can be engineered into the proteinbetween the tag and the fusion itself to facilitate removal of the tagafter purification.

Commercially produced protein expression/purification kits providetailored protocols for the purification of proteins made using eachsystem. See, for instance, the QIAexpress™ expression system from QIAGEN(Chatsworth, Calif.) and various expression systems provided byINVITROGEN (Carlsbad, Calif.). Where a commercial kit is employed toproduce a bispecific fusion protein, the manufacturer's purificationprotocol is a preferred protocol for purification of that protein. Forinstance, proteins expressed with an amino-terminal hexa-histidine tagcan be purified by binding to nickel-nitrilotriacetic acid (Ni-NTA)metal affinity chromatography matrix (The QIAexpressionist, QIAGEN,1997).

Alternately, the binding specificities of the cell-binding/targetingdomain of the disclosed apoptosis-modifying protein may be exploited tofacilitate specific purification of the proteins. A preferred method ofperforming such specific purification would be column chromatographyusing column resin to which the target cell surface receptor, or anappropriate epitope or fragment or domain of the target molecule, hasbeen attached.

If the apoptosis-modifying fusion protein is produced in a secretedform, e.g. secreted into the milk of a transgenic animal, purificationwill be from the secreted fluid. Alternately, purification may beunnecessary if it is appropriate to apply the fusion protein directly tothe subject in the secreted fluid (e.g. milk).

III. Variation of a Bispecific Fusion Protein

A. Sequence Variants

The binding and apoptosis-modifying characteristics of theapoptosis-modifying fusion proteins disclosed herein lies not in theprecise amino acid sequence, but rather in the three-dimensionalstructure inherent in the amino acid sequences encoded by the DNAsequences. It is possible to recreate the functional characteristics ofany of these proteins or protein domains of this invention by recreatingthe three-dimensional structure, without necessarily recreating theexact amino acid sequence. This can be achieved by designing a nucleicacid sequence that encodes for the three-dimensional structure, butwhich differs, for instance by reason of the redundancy of the geneticcode. Similarly, the DNA sequence may also be varied, while stillproducing a functional apoptosis-modifying fusion protein.

Variant apoptosis-modifying fusion proteins include proteins that differin amino acid sequence from the disclosed sequence but that sharestructurally significant sequence homology with any of the providedproteins. Variation can occur in any single domain of the fusion protein(e.g., the binding or apoptosis-modifying domain, or, where appropriate,the linker). Variation can also occur in more than one of such domainsin any particular variant protein. Such variants may be produced bymanipulating the nucleotide sequence of, for instance, aBcl-x_(L)-encoding sequence, using standard procedures, such assite-directed mutagenesis or PCR. The simplest modifications involve thesubstitution of one or more amino acids for amino acids having similarbiochemical properties. These so-called conservative substitutions arelikely to have minimal impact on the activity of the resultant protein,especially when made outside of the binding site or active site of therespective domain. The regions or sub-domains of DTR that are essentialto targeted cell binding are known in the art (see, Choe et al., Nature357:216-222, 1992; Parker and Pattus, TIBS 18:391-395, 1993). Regions orsub-domains of Bcl-2 proteins responsible for apoptosis modification areunder intense study; much of this work is reviewed in Adams and Cory,Science 281:1322-1326.

Table 1 shows amino acids that may be substituted for an original aminoacid in a protein, and which are regarded as conservative substitutions.

TABLE 1 Original Residue Conservative Substitutions Ala ser Arg lys Asngln; his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu;val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

More substantial changes in protein structure may be obtained byselecting amino acid substitutions that are less conservative than thoselisted in Table 1. Such changes include changing residues that differmore significantly in their effect on maintaining polypeptide backbonestructure (e.g., sheet or helical conformation) near the substitution,charge or hydrophobicity of the molecule at the target site, or bulk ofa specific side chain. The following substitutions are generallyexpected to produce the greatest changes in protein properties: (a) ahydrophilic residue (e.g., seryl or threonyl) is substituted for (or by)a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl oralanyl); (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain (e.g.,lysyl, arginyl, or histadyl) is substituted for (or by) anelectronegative residue (e.g., glutamyl or aspartyl); or (d) a residuehaving a bulky side chain (e.g., phenylalanine) is substituted for (orby) one lacking a side chain (e.g., glycine).

Variant binding domain, apoptosis-modifying domain, or fusionprotein-encoding sequences may be produced by standard DNA mutagenesistechniques, for example, M13 primer mutagenesis. Details of thesetechniques are provided in Sambrook et al., In Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 15. By the use ofsuch techniques, variants may be created which differ in minor ways fromthe apoptosis-modifying fusion protein-encoding sequences disclosed. DNAmolecules and nucleotide sequences which are derivatives of thosespecifically disclosed herein and that differ from those disclosed bythe deletion, addition, or substitution of nucleotides while stillencoding a protein that binds to a target cell, translocates orotherwise integrates into the target cell membrane(s), and therebymodifies an apoptotic response in the target cell, are comprehended bythis invention. In their most simple form, such variants may differ fromthe disclosed sequences by alteration of the coding region to fit thecodon usage bias of the particular organism into which the molecule isto be introduced.

Alternatively, the coding region may be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed fusion sequences. For example,the 57th amino acid residue of the Bcl-x_(L)-DTR protein is alanine. Thenucleotide codon triplet GCC encodes this alanine residue. Because ofthe degeneracy of the genetic code, three other nucleotide codontriplets—(GCG, GCT and GCA)—also code for alanine. Thus, the nucleotidesequence of the disclosed Bcl-x_(L)-DTR encoding sequence could bechanged at this position to any of these three alternative codonswithout affecting the amino acid composition or characteristics of theencoded protein. Based upon the degeneracy of the genetic code, variantDNA molecules may be derived from the cDNA and gene sequences disclosedherein using standard DNA mutagenesis techniques as described above, orby synthesis of DNA sequences. Thus, this invention also encompassesnucleic acid sequences which encode an apoptosis-modifying fusionprotein, but which vary from the disclosed nucleic acid sequences byvirtue of the degeneracy of the genetic code. Apoptosis assays,including those discussed herein, can be used to determine the abilityof the resultant variant protein to modify apoptosis.

B. Peptide Modifications

The present invention includes biologically active molecules that mimicthe action of the apoptosis-modifying fusion proteins of the presentinvention, and specifically modify apoptosis in a target cell. Theproteins of the invention include synthetic versions ofnaturally-occurring proteins described herein, as well as analogues(non-peptide organic molecules), derivatives (chemically functionalizedprotein molecules obtained starting with the disclosed peptidesequences) and variants (homologs) of these proteins that specificallybind to a chosen target cell and modify apoptosis in that target cell.Each protein of the invention is comprised of a sequence of amino acids,which may be either L- and/or D-amino acids, naturally occurring andotherwise.

Proteins may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedproteins, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the protein,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the protein side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the protein side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the protein sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the proteins of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are also within the scopeof the present invention, whereby the three-dimensional arrangement ofthe chemical constituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the protein backbone and componentamino acid side chains in the apoptosis-modifying fusion protein,resulting in such peptido- and organomimetics of the proteins of thisinvention having measurable or enhanced neutralizing ability. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, Computer-AssistedModeling of Drugs, in Klegerman & Groves (eds.), PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., 165-174, 1993; andMunson (ed.) Principles of Pharmacology, Ch. 102, 1995, for descriptionsof techniques used in CADD. Also included within the scope of theinvention are mimetics prepared using such techniques that produceapoptosis-modifying fusion proteins.

IV. Activity of Fusion Proteins

Because the apoptosis modifying fusion proteins provided in thisinvention are at least bi-functional, having one domain required forcell targeting and another for modification of apoptosis in the targetcell, there are at least two activities for each fusion protein. Theseinclude the affinity of the fusion protein for a specific target cell,class of target cells, tissue type, etc., (the binding ability), and theability of the targeted fusion to effect apoptosis in the targeted cell(the apoptosis-modifying ability). Various techniques can be used tomeasure each of these activities.

A. Fusion Protein Affinity for Target Cells Fusion protein affinity forthe target cell, or to a specific cell surface protein, can bedetermined using various techniques known in the art. One common methodis a competitive binding assay (Greenfield et al., Science 238:536-539,1987). In a competitive binding assay, radiolabeled receptor bindingprotein, or a derivative or fragment thereof, is exposed to the targetnative cell in the presence of one or varying concentrations of coldfusion protein and other competitive proteins being assayed. The amountof bound, labeled binding protein can be measured through standardtechniques to determine the relative cell-binding affinity of thefusion.

B. Apoptosis Inhibition or Enhancement

Several in vitro systems are used to study the process of apoptosis.These include growth factor deprivation in culture, treatment of cellswith staurosporine (a non-specific protein kinase inhibitor),application of γ-radiation, and infection by viruses. Apoptosis asstimulated by any signal can be examined or measured in a variety ofways. Detection of morphological indicia of apoptosis (e.g., membraneblebbing, chromatin condensation and fragmentation, and formation ofapoptotic bodies) can provide qualitative information. More quantitativetechniques include TUNEL staining, measurement of DNA laddering,measurement of known caspase substrate degradation (e.g., PARP; Tayloret al., J. Neurochem. 68:1598-605, 1997) and counting dying cells, whichhave become susceptible to dye uptake. Many companies (e.g., Trevigen,Gaithersburg, Md.; and R&D Systems, Minneapolis, Minn.) also supply kitsuseful for the measurement of apoptosis by various methods; many ofthese kits can be used to measure the effect of disclosedapoptosis-modifying fusion proteins on apoptosis in a variety of celltypes.

By way of example, the following techniques can be used to measure themodification of apoptosis caused in a target cell after it is contactedwith an apoptosis-modifying fusion protein of the present invention.

TUNEL staining: Terminal end-labeling of broken DNA fragments withlabeled nucleotides; the reaction is catalyzed by terminal nucleotidetransferase (TdT). Various kits are available for measurement of TUNELstaining, including the TdT in situ TUNEL-based Kit (R&D Systems,Minneapolis, Minn.).

Measurement of Caspase Activity: Another common system for measuring theamount of apoptosis occurring in an in vitro cell system is to measurethe poly-ADP ribose Polymerase (PARP) cleavage after treatment of thecells with various stimulators of apoptosis. PARP is a known substratefor a caspase (CPP-32) involved in the apoptotic kinase cascade. Thistechnique can be carried out using essentially the following protocol.HeLa cells are plated in growth media (e.g., EMEM containing 10% FBS at2×10⁵ cells/ml) and treated with one or more concentrations of anapoptosis-modifying fusion protein according to the current invention.The appropriate concentration for each fusion protein will depend onvarious factors, including the fusion protein in question, target cell,and apoptosis stimulator employed. Appropriate concentrations mayinclude, for instance, about 0.5 μM to about 3 μM final. It may bebeneficial to treat the target cells multiple times with the fusionprotein, usually after a period of incubation ranging from one toseveral hours. For instance, cells can be exposed to the fusion proteina second time about fifteen hours after the original treatment. Usuallythe same concentration(s) of fusion protein is used in the secondtreatment.

Apoptosis is induced immediately after the last treatment of the targetcells with apoptosis modifying fusion protein. The method of applicationof the apoptosis stimulus, amount applied, appropriate incubation timewith the inducer, etc., will be specific to the type of apoptosisinduction used (e.g., staurosporine, γ-radiation, virions, caspaseinhibitor, etc.). Such details are in general well known to those ofordinary skill in the art. After an appropriate incubation period, celllysates are prepared from the treated target cells, and aliquots loadedonto SDS-PAGE for analysis. The resultant gels can be examined using anyof various well-known techniques, for instance by performing a Westernanalysis immunoblotted with anti-PARP polyclonal antibody (BoehringerMannheim GmbH, Germany), developed with enhanced chemiluminescence.

Known inhibitors of apoptotic pathways, for instance caspase inhibitors,can be used to compare the effectiveness of apoptosis-modifying fusionproteins of this invention. Appropriate inhibitors include viral caspaseinhibitors like crmA and baculovirus p35, and peptide-type caspaseinhibitors including zVAD-fmk, YVAD- and DEVD-type inhibitors. SeeRubin, British Med. Bulle., 53:617-631, 1997.

V. Incorporation of Apoptosis-Modifying Fusion Proteins intoPharmaceutical Compositions

Pharmaceutical compositions that comprise at least one apoptosismodifying fusion protein as described herein as an active ingredientwill normally be formulated with an appropriate solid or liquid carrier,depending upon the particular mode of administration chosen. Thepharmaceutically acceptable carriers and excipients useful in thisinvention are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, other proteins,such as human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

One or more other medicinal and pharmaceutical agents, for instancechemotherapeutic, anti-inflammatory, anti-viral or antibiotic agents,also may be included.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical and oral formulations can be employed.Topical preparations can include eye drops, ointments, sprays and thelike. Oral formulations may be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powders, pills, tablets, or capsules). Forsolid compositions, conventional non-toxic solid carriers can includepharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

The pharmaceutical compositions that comprise apoptosis modifying fusionprotein will preferably be formulated in unit dosage form, suitable forindividual administration of precise dosages. One possible unit dosagecontains approximately 100 μg of protein. The amount of active compoundadministered will be dependent on the subject being treated, theseverity of the affliction, and the manner of administration, and isbest left to the judgment of the prescribing clinician. Within thesebounds, the formulation to be administered will contain a quantity ofthe active component(s) in an amount effective to achieve the desiredeffect in the subject being treated. Ideally, a sufficient amount of theprotein is administered to achieve tissue a concentration at the site ofaction that is at least as great as in vitro concentrations that havebeen shown to be effective.

VI. Clinical Use of Apoptosis-Modifying Fusion Proteins

The targeted apoptosis-regulating activity exhibited by the disclosedfusion proteins makes these fusions useful for treatingneurodegenerative diseases, transient ischemic injuries, and unregulatedcell growth (as may for instance be found in tumors and variouscancers).

The apoptosis-modifying fusion proteins of this invention may beadministered to humans, or other animals on whose cells they areeffective, in various manners such as topically, orally, intravenously,intramuscularly, intraperitoneally, intranasally, intradermally,intrathecally, and subcutaneously. Administration of apoptosis-modifyingfusion protein composition is indicated for patients with aneurodegenerative disease, suffering from stroke episodes or transientischemic injury, or experiencing uncontrolled or unwanted cell growth,such as malignancies or neoplasms. More generally, treatment isappropriate for any condition in which it would be beneficial to alter(either inhibit or enhance) an apoptotic response of a subject's targetcells. The particular mode of administration and the dosage regimen willbe selected by the attending clinician, taking into account theparticulars of the case (e.g., the patient, the disease, and thedisease-state involved). By way of example, when apoptosis is beinggenerally inhibited over the short term, for instance after transientischemic neuronal injury, it may be advantageous to administerrelatively large doses of fusion protein repeatedly for a few days. Incontrast, if apoptosis is being enhanced in specific cell types, forinstance in hyper-proliferative cells, it may be of greater benefit toapply a relatively small dose of fusion protein repeatedly, e.g., daily,weekly, or monthly, over a much longer period of treatment.

In addition to their individual use, apoptosis-modifying fusion proteinsas disclosed in the current invention may be combined with varioustherapeutic agents. For instance, an apoptosis-enhancing fusion proteinsuch as Bad-DTTR may be combined with or used in association with otherchemotherapeutic or chemopreventive agents for providing therapy againstneoplasms or other hyper-proliferative cellular growth conditions.Various such anti-cancer agents are well known to those of ordinaryskill in the art. Apoptosis-modifying fusion proteins according to thisinvention also can be supplied in the form of kits; the construction ofkits appropriate for therapeutically active proteins known.

EXAMPLE 1 Construction of Functional Apoptosis-Modifying Fusion ProteinsA. Bcl-x_(L)-DTR

The human Bcl-x_(L) gene from codon 1 through 233 (provided by Dr. CraigThompson) and the diphtheria toxin gene from codon 384 through 535(receptor binding domain, DTR), containing mutations in codons 508 and525, were amplified by PCR so that the DT mutation at codon 525 wasmutated to the wild-type by the PCR primer. The two PCR products,Bcl-x_(L)1-233 and DT384-535 (DTR), were digested with NdeI/NotI andNotI/XhoI restriction enzymes, respectively. Bcl-x_(L) was fused to the5′ end of the DTR gene with a linker (GCG TAT TCT GCG GCC GCG, SEQ IDNO: 5) to encode for Ala Tyr Ser Ala Ala Ala (SEQ ID NO: 6) between thetwo peptide domains. The two digested fragments were ligated into theprokaryotic expression vector pET16b (Novagen, Inc., Madison, Wis.) cutwith NdeI and XhoI (FIG. 1A). The codon 508 of DTR was mutated to thewild-type form (Phe→Ser) and the first three nucleotides (CAT) of NdeIwere deleted by double-stranded, site-directed mutagenesis. FIG. 1Ashows a schematic representation of the resultant apoptosis-modifyingfusion protein, Bcl-x_(L)-DTR.

As controls, human Bcl-x_(L) (codons 1-233) and DTR (codons 384-535 ofDT) genes were separately subcloned into pET16b vectors through NdeI andXhoI sites. The histidine tag and Factor Xa digestion site sequencesfrom the expression vector were upstream of Bcl-x_(L), DTR andBcl-x_(L)-DTR coding sequences. All three expression constructs wereverified by sequencing.

For expression in eukaryotic cells, Bcl-x_(L)-DTR and Bcl-x_(L) geneconstructs were inserted in the eukaryotic vector pcDNA3 (Invitrogen,Carlsbad, Calif.) and the constructs verified by sequencing.

B. Bad-DTTR

The full-length mouse Bad gene with two Ser→Ala mutations at codons 112and 136 (Schendel et al., Proc. Natl. Acad. Sci. USA 94:5113-5118,1997), and the diphtheria toxin gene from codons 194 through 535(translocation and receptor-binding domains, DTTR, without the catalyticdomain) were amplified by PCR. The two PCR products, Bad and DT194-535(DTTR), were used as templates to directly fuse the Bad gene to the 5′end of DTTR gene by a second round of PCR. The Bad-DTTR gene fragmentwas digested with NdeI and XhoI and ligated into the prokaryoticexpression vector pET16b (Novagen, Inc., Madison, Wis.) digested withNdeI and XhoI. The histidine tag and Factor Xa digestion site sequencesfrom the expression vector were upstream of the Bad-DTTR codingsequence. The expression construct was verified by sequencing.

EXAMPLE 2 Expression and Purification of Functional Apoptosis-ModifyingFusion Proteins A. Prokaryotic Expression

To produce proteins for extracellular addition to cells, the Bcl-x_(L)gene, the DTR domain gene and the Bcl-x_(L)-DTR fusion gene were clonedinto pET16b. E. coli BL21(DE3) strain was used to express Bcl-x_(L)-DTR,Bad-DTTR, Bcl-x_(L) and DTR, with addition of 1 mM IPTG when the OD260reached 0.5-0.7. After two hours incubation and lysis by French pressthe inclusion bodies were collected and dissolved in 6M guanidine-HCl.

B. Eukaryotic Expression

Transfection of HeLa cells with the fusion constructs was performed asreported previously (Wolter et al., J Cell Biol 139:1281-1292, 1997).HeLa cells were harvested and lysed in 1 ml buffer containing 100 μg/mlleupeptin 20 hours after transfection, centrifuged to remove celldebris, and 15 μl aliquots of the supernatant loaded onto 10-20%SDS-PAGE. The plasmid encoded proteins were visualized by immunoblottingwith anti-Bcl-x_(L) monoclonal antibody (2H12, Trevigen, Gaithersburg,Md.) and developed using enhanced chemiluminescence (Amersham Inc.,Arlington Heights, Ill.). Results are shown in FIG. 1B.

C. Purification

Histidine tag binding resin (Novagen, Inc., Madison, Wis.) was used topurify Bcl-x_(L)-DTR, Bad-DTTR, Bcl-x_(L), and DTR. Proteins wererefolded by dialysis against, or dilution into, 100 mM Tris-Acetate (pH8.0)/0.5 M arginine, concentrated with PEG15,000-20,000 and dialyzedagainst PBS. This yielded protein purified to greater than 90%homogeneity. The four proteins were subjected to 10-20% SDS-PAGE,visualized by immunoblotting with either anti-Bcl-x_(L) monoclonal(2H12) or horse anti-DT polyclonal antibodies (Centers for DiseaseControl, Atlanta, Ga.) and developed as above. They were of the expectedmolecular weight on SDS PAGE and of the expected immunoreactivity toantibodies against Bcl-x_(L) or DT on Western blots.

EXAMPLE 3 Assays for Measuring Fusion Protein Binding to, andTranslocation into, Target Cells A. Competitive Binding Assay

Protein binding to the diphtheria toxin receptor was performed aspreviously reported (Greenfield et al., Science 238:536-539, 1987) withthe following modifications. DT was radiolabeled with I¹²⁵ usingiodobeads (Pierce Chem. Co., Rockford, Ill.) as described by themanufacturer. Cos-7 cells, grown to confluency in 12 well costar plates,were analyzed for receptor binding and competition by incubation forthree hours on ice. Results are reported in FIG. 2. Cold competitorproteins, native DT (Δ), Bcl-x_(L)-DTR (▴), Bcl-x_(L) (◯), and DTR (),were used to displace I¹²⁵ labeled DT tracer.

Native DT and Bcl-x_(L)-DTR compete for DT receptor binding in thenanomolar concentration range. DT and the Bcl-x_(L)-DTR fusion proteincompeted for I¹²⁵-DT binding to its receptor to a similar extentalthough the affinity of the fusion was three times lower than that ofnative DT (FIG. 2). Neither the Bcl-x_(L) domain alone nor the DTRdomain alone was able to compete for DT receptor binding. The morecomplete protein (Bcl-x_(L)-DTR), where Bcl-x_(L) is substituted for theDT translocation domain, folded such that DT receptor binding activitywas retained whereas the isolated binding domain (DTR) did not. Additionof the DT A chain domain to the N-terminus of Bcl-x_(L)-DTR furtherincreased the affinity of the chimera to the DT receptor.

B. Assays for Effective Transport of the Fusion Protein into the TargetCell

Diphtheria toxin is endocytosed by cells and reaches low pHintracellular compartments. The low pH triggers a conformational changein the translocation domain, which allows this domain to insert intomembranes and form channels. The toxicity of DT is blocked bylysosomotropic agents such as chloroquine, which increase the pH ofintracellular compartments. Chloroquine at a concentration that blocksdiphtheria toxin toxicity (10 μM) did not block the activity ofBcl-x_(L)-DTR to inhibit poliovirus-induced cell death. Thus, themechanism of membrane interaction of Bcl-x_(L)-DTR differs to someextent from that of DT. However, brefeldin A, an inhibitor of vesicletraffic between the ER and the Golgi apparatus (Lippincott-Schwartz etal., Cell 67:601-616, 1991; Hunziker et al., Cell 67:617-627, 1991),does block the anti-apoptosis activity of Bcl-x_(L)-DTR (Table 3). Theseresults indicate that Bcl-x_(L)-DTR must be endocytosed and suggest thatBcl-x_(L)-DTR must reach the Golgi apparatus or the ER to prevent celldeath. The subcellular location from which native Bcl-2 family membersregulate apoptosis is currently under scrutiny (Hunziker et al., Cell67:617-627, 1991). Several intracellular membrane locations, includingthe ER, appear able to mediate Bcl-2 family regulation of cell death(Krajewski et al., Cancer Res. 53:4701-4714, 1993). Bcl-x_(L)-DTR mayreach the ER to translocate into the cell cytosol or perhapsBcl-x_(L)-DTR, when bound closely to a membrane, can insert into thatmembrane and inhibit apoptosis in the membrane-intercalated form.

EXAMPLE 4 Measurement of Bcl-x_(L)-DTR Apoptosis-Inhibiting Activity

A. Apoptosis Inhibition after Transient Cell Transfection

To demonstrate that Bcl-x_(L)-DTR is effective at inhibiting apoptosiswhen expressed from within the target cell, this construct and thecontrol construct containing Bcl-x_(L) were transiently transfected intoHeLa cells. Assay of apoptosis inhibition after transient transfectionwas performed as reported previously (Wolter et al., J. Cell Biol.139:1281-1292, 1997). The Bcl-x_(L)-DTR fusion gene blocked apoptosisafter transient transfection into HeLa cells (FIG. 1C) to an extentsimilar to that of the Bcl-x_(L) gene after C-terminal tail truncation(Wolter et al., J Cell Biol 139:1281-1292, 1997).

B. Inhibition of STS-Induced Apoptosis by Extracellular Treatment withBcl-x_(L)-DTR

Hoechst dye no. 33342 staining: The effectiveness of extracellulardelivery of Bcl-x_(L) or the Bcl-x_(L)-DTR fusion protein for inhibitingthe rate of cell death by apoptosis was examined as follows. Cos-7 cellsat 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBS were incubated with 0.1μM STS (◯), 0.1 μM STS plus 4.8 μM Bcl-x_(L)-DTR protein added to themedium (Δ) or 20 μl of PBS (◯). Apoptotic cells were quantified bystaining with Hoechst dye no. 33342. Results in FIG. 3A are presented asthe average number of cells per field (magnification 160×). For eachpoint, at least 5 fields were counted in each of at least 3 wells.Bcl-x_(L)-DTR dramatically decreased the rate of apoptosis in Cos-7cells. Six different preparations of Bcl-x_(L)-DTR were found to haveactivity and the apoptosis prevention activity was stable for at least 5months when Bcl-x_(L)-DTR was stored at 4° C. Addition of Bcl-x_(L)-DTRminutes before the addition of STS blocked more than 70% of Cos-7 celldeath after 6 hours and more than 50% of cell death after 12 hours ofSTS exposure (FIG. 3A).

Jurkat, HeLa and U251 cells were also protected from STS-inducedapoptosis by Bcl-x_(L)-DTR (Table 2). Bcl-x_(L) protein added to Cos-7cells, however, did not alter the extent of cell death induced by STS. Anontoxic DT mutant able to bind the DT receptor, CRM197, also had noeffect on apoptosis induced by STS. To further test the role of DTreceptor binding in apoptosis inhibition, cells expressing DT receptorswere compared with cells lacking DT receptors. Mouse and rat cells arethousands of times less sensitive to DT than human or monkey cell linesdue to a lack of the DT receptor (Pappenheimer The Harvey Lectures76:45-73, 1982). Comparing human, monkey, mouse and rat cell linesrevealed that those cells lacking the DT receptor, WEHI-7.1 and 9L, wereinsensitive to apoptosis protection by Bcl-x_(L)-DTR (Table 2). Thesensitivity of the six cell lines to DT toxicity, thought to reflect DTreceptor levels, correlated with sensitivity to apoptosis prevention byBcl-x_(L)-DTR (Table 2).

The magnitude of apoptosis inhibition by extracellular Bcl-x_(L)-DTR(FIG. 3A, Table 2) was similar to that found by transfection of thefusion gene into cells (FIG. 1C). Although fusion to the C-terminus ofBcl-x_(L) inhibited bioactivity relative to native Bcl-x_(L) aftertransfection (FIG. 1C), a very substantial prevention of cell death wasobtained at both the gene level and the protein level (FIG. 3A). Thusthe delivery of Bcl-x_(L)-DTR is efficient and apoptosis can beprevented by delivery of Bcl-x_(L) from the outside of cells.

Measurement of caspase activity: To confirm the results of cell deathmeasurements by Hoechst staining and trypan blue dye exclusion, weexamined caspase-induced cleavage of poly-ADP ribose polymerase (PARP).HeLa cells were plated in EMEM containing 10% FBS at 2×10⁵ cells/ml andtreated with two different preparations of Bcl-x_(L)-DTR at 1.48 μM or 1μM. Fifteen hours later, cells were treated again with Bcl-x_(L)-DTR at1.48 μM or 1 μM. Immediately after the second treatment, 0.8 μM STS wasadded. Three hours later, cell lysates were made and aliquots wereloaded onto SDS-PAGE, immunoblotted with anti-PARP polyclonal antibody(Boehringer Mannheim GmbH, Germany) and developed with enhancedchemiluminescence. Lane a contains control HeLa cells not incubated withSTS (uninduced cells); Lane b, HeLa cells treated with STS plus 1 μMBcl-x_(L)-DTR protein; Lane c, HeLa cells treated with STS plus 1.48 μMBcl-x_(L)-DTR protein; and Lane d, HeLa cells treated with STS and nofusion protein. HeLa cells incubated with Bcl-x_(L)-DTR showedsignificantly less cleavage of PARP after apoptosis induction with STS(FIG. 3B).

C. Inhibition of γ-Radiation-Induced Apoptosis by ExtracellularTreatment with Bcl-x_(L)-DTR

Radiation is a potent inducer of apoptosis in many hematopoetic celltypes. The ability of Bcl-x_(L)-DTR to prevent radiation-inducedapoptosis was examined in the human T cell line, Jurkat. When added tothe media (serum-free RPMI-1640 medium with insulin and transferrin) ofJurkat cells plated at 10⁵ cells/ml a few minutes prior to induction ofapoptosis by 10 gray γ-radiation, Bcl-x_(L)-DTR (4.63 μM) blocked almosthalf of the ensuing cell death (FIG. 4A). Apoptotic cells were countedusing Hoechst dye no. 33342. Control cells were not irradiated and nottreated with Bcl-x_(L)-DTR.

In a clonogenic assay measuring long term survival, Jurkat cells showedmore than a 3-fold greater survival when Bcl-x_(L)-DTR was added to themedia immediately prior to 5 gray γ-radiation.

D. Inhibition of Anti-Fas-Induced Apoptosis by Extracellular Treatmentwith Bcl-x_(L)-DTR

Jurkat cells are also sensitive to apoptosis induced by antibody bindingto the Fas/APO-1/CD95 receptor. The Fas pathway of apoptosis is one ofthe few pathways shown to be less sensitive or insensitive to apoptosisprotection by Bcl-2 and Bcl-x_(L) (Boise & Thompson, Proc. Natl. Acad.Sci. USA, 94:3759-3764, 1997; Memon et al., J. Immunol. 155:4644-4652,1995) and contrasts with radiation-induced apoptosis in this regard.Jurkat cells were plated at 10⁵ cells/ml in serum-free RPMI-1640 mediumwith insulin and transferrin, and treated with 100 ng/ml anti-Fasantibody (CH11, Upstate Biotechnology, Lake Placid, N.Y.) minutes afteraddition of Bcl-x_(L)-DTR to a concentration 4.68 μM. Control cells weretreated with PBS and no anti-Fas antibody. Fas antigen-induced apoptosis(measured by counting dying cells using Hoechst dye no. 33342) showedvery little inhibition by Bcl-x_(L)-DTR, although there was astatistically significant decrease in apoptosis between 2 and 4 hours insome experiments (FIG. 4B). The degree of protection of differentapoptosis pathways by extracellular Bcl-x_(L)-DTR corresponded with thatseen by transfection with the Bcl-x_(L) gene.

E. Inhibition of Poliovirus-Induced Apoptosis by Extracellular Treatmentwith Bcl-x_(L)-DTR

Viruses induce a powerful apoptosis response in certain cells andprevention of this apoptosis may have therapeutic utility (Hardwick,Adv. Pharm. 41:295-336, 1997). Poliovirus-induced apoptosis of HeLacells was also examined for sensitivity to extracellular Bcl-x_(L)-DTR,a system where inhibition of cell death by transfection with theBcl-x_(L) gene has been demonstrated (Castelli et al., J Exp. Med.186:967-972, 1997). Adding Bcl-x_(L)-DTR 30 minutes after infection ofcells with low titers (MOI of 1 pfu/cell) of poliovirus (FIG. 5) or withmoderately high titers (MOI of 20 pfu/cell) of poliovirus prevented morethan half of the cell death for up to 24 hours. Addition ofextracellular Bcl-x_(L) or the DTR domain proteins alone had no affecton poliovirus-induced apoptosis.

F. Competition of Apoptosis Inhibition

Caspase inhibitors block many pathways of apoptosis and are beingexplored for pharmacologic potential to inhibit cell death (Chen et al.,Nature 385:434-439, 1997). zVAD-fmk and Boc-D-fmk are powerful, broadspecificity caspase inhibitors that block many apoptosis pathways(Henkart, Immunity 4:195-201, 1996). Apoptosis inhibition activity ofzVAD-fmk and Boc-D-fmk was compared with that of Bcl-x_(L)-DTR. HeLacells were plated at a density of 1×10⁵ cells/well in EMEM containing10% FBS and antibiotics, infected with poliovirus at an MOI of 1pfu/cell as reported previously (Castelli et al., J Exp Med 186:967-972,1997) and immediately treated with negative control peptide zFA-fmk at20 μM, Bcl-x_(L)-DTR at 0.48 μM, or peptides zVAD-fmk or Boc-D-fmk at 20μM. Cell viability was assessed by trypan blue dye exclusion 24 hoursfollowing addition of virus. zFA-fmk, zVAD-fmk and Boc-D-fmk were fromEnzyme Systems Products, Dublin, Calif.

Bcl-x_(L)-DTR at 0.48 μM blocked cell death to a greater extent thaneither zVAD-fmk or Boc-D-fmk at 20 μM (FIG. 5). Bcl-x_(L)-DTR showed astrong inhibition of a potent and pathologically important apoptosispathway. Interestingly, Bcl-x_(L) appears to act at an early step in thecell death pathway when intervention can permit long term viability ofcells, whereas caspase inhibitors appear to work relatively moredownstream in the apoptosis pathway (Chinnaiyan et al., J Biol Chem271:4573-4576, 1996; Xiang et al., Proc. Natl. Acad. Sci. USA93:14559-14563, 1996; Miller et al., J. Cell Biol 139:205-217, 1997).

EXAMPLE 5 Measurement of Bad-DTTR Apoptosis-Enhancing Activity

A. Stimulation of Apoptosis by Extracellular Treatment with Bad-DTTR

To determine the effectiveness of the fusion protein Bad-DTTR attriggering apoptosis, cell survival after exposure to Bad-DTTR wasexamined. U251 MG cells at 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBSwere incubated with 0.65 μM Bad-DTTR protein added to the medium or 20μl of PBS. Total and apoptotic cells were quantified by staining withHoechst dye no. 33342. Results are presented in FIG. 6 as the averagenumber of cells per field (magnification 160×). Bad-DTTR decreases cellviability 12 hours after treatment.

B. Enhancement of STS-Triggered Apoptosis by Extracellular Treatmentwith Bad-DTTR

To examine the ability of Bad-DTTR to enhance apoptosis triggered bySTS, cell survival was determined after exposure to variousconcentrations of STS, in combination with various combinations ofBad-DTTR. U251 MG cells at 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBSwere treated with PBS, 0.1 μM STS, 0.65 μM Bad-DTTR, 0.065 μM Bad-DTTR,0.1 μM STS plus 0.65 μM Bad-DTTR and 0.1 μM STS plus 0.065 μM Bad-DTTR.Apoptotic death cells were quantified at different times by stainingwith Hoechst dye no. 33342. Results are presented as the average numberof cells per field (magnification 160×). Apoptosis is most enhanced whencells are treated with 0.1 μM STS plus 0.65 μM Bad-DTTR, and cells beginto die about 12 hours after treatment.

U251 MG cells at 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBS weretreated with PBS, 1 μM STS, 0.65 μM Bad-DTTR, 0.065 μM Bad-DTTR, 1 μMSTS plus 0.65 μM Bad-DTTR and 1 μM STS plus 0.065 μM Bad-DTTR. Apoptoticcells were quantified and presented as above. The combination of 1 μMSTS and Bad-DTTR at various concentrations causes an earlier onset ofapoptosis in U251 MG cells.

EXAMPLE 6 LF_(n)-Bcl-x_(L) Inhibits Neuron, Macrophage, and LymphocyteApoptosis

Anthrax toxin includes three components: lethal factor (LF), edemafactor (EF) and protective antigen (PA) (Leppla, Anthrax toxin. InHandbook of Natural Toxins, Moss et al., Eds., Dekker, New York, Vol. 8,pp. 543-572, 1995). PA binds simultaneously to LF and to a cell surfacereceptor existing on the cells of almost all species including rodents(Leppla, 1995; Friedlander, J. Biol. Chem. 261:7123-7126, 1986), andtransports LF into cells where LF causes toxic effects. PA alone,however, is not toxic. It has been found that the first 255 residues(LF_(n)) of LF, which constitute the PA-binding domain and are not toxicto cells, are sufficient for delivery of heterologous peptides to thecytosol. Cytotoxins have been fused to LF_(n) (Leppla, 1995; Arora etal., J Biol. Chem. 269:26165-26171, 1994; Milne et al., Mol. Microbiol.15: 661-666, 1995). Administration of a fusion protein containing LF_(n)and the gp120 envelope glycoprotein of HIV-1 along with PA toantigen-presenting cells sensitized them to cytolysis by cytotoxicT-lymphocytes (CTL) specific to gp120 (Goletz et al., Proc Natl Acad SciUSA 94:12059-12064, 1997). In vivo, LF_(n)-fused to CTL epitopesinjected along with PA has been shown to stimulate a CTL responseagainst the antigens in mice (Ballard et al., Proc. Natl. Acad. Sci. USA93: 12531-12534, 1996; Ballard et al, Infect. Immun. 66:615-619, 1998;Ballard et al., Infect. Immun. 66:4696-4699, 1998; Doling et al.,Infect. Immun. 67: 3290-3296, 1999).

To inhibit neuron apoptosis, another protein delivery system wasengineered by fusing a nontoxic domain of anthrax toxin to Bcl-x_(L), tocreate the LF_(n)-Bcl-x_(L) chimeric fusion protein. Macrophage andlymphocyte death in culture, and neuron death in vivo in a retinalganglion cell model of apoptosis induced by axotomy, can be prevented byapplication of this fusion protein.

A. Construction of LF_(n)-Bcl-x_(L) in a Prokaryotic Expression Plasmid

The coding sequence for lethal factor (LF) from codons 34 to 288(LF_(n)) (Bragg et al., Gene 81:45-54, 1989), which is theamino-terminal domain (residues 1-255) of mature LF (Leppla, 1995), wasamplified using PCR with the template of pET15b/LF_(n) (Milne et al.,Mol. Microbiol. 15: 661-666, 1995). The gene of human Bcl-x_(L) fromCodons 1 to 209 (Bcl-x_(L)(1-209)) (Boise et al., Cell 74: 597-608,1993) was amplified by PCR. Then the LF_(n) encoding sequence was fusedto the 5′ end of Bcl-x_(L)(1-209) encoding sequence by a second round ofPCR. A stop codon was introduced immediately after Codon 209 ofBcl-x_(L). The fused DNA fragment, LF_(n)-Bcl-x_(L), was cut with NdeIand Xho I, and inserted into prokaryotic expression vector pET15b cutwith Nde I and Xho I (FIG. 8). A histidine tag and thrombin cleavagesite were linked to the N-terminal of LF_(n)-Bcl-x_(L). Similarly, theBcl-x_(L) gene from codons 1 to 209 was also genetically inserted intopET15b at the sites of Nde I and Xho I. All the constructs were verifiedby DNA sequencing.

B. Construction of Eukaryotic Expression Plasmids, Transfection, WesternBlotting and Biologic Activity Assay

The sequences encoding LF_(n)-Bcl-x_(L), Bcl-x_(L) from codons 1 to 209,and full-length Bcl-x_(L), were separately engineered into eukaryoticexpression vector pcDNA3.1+ and verified by DNA sequencing. Cos-7 cellswere co-transfected with plasmid EGFP-C3 and one of the three plasmidsas reported (Keith et al., J Cell Biol 139: 1281-1292, 1997). The cellswere treated with 0.1 μM staurosporine (STS) 12 hours later. The deadand living cells were counted with Hoechst 33342 at different timesafter STS treatment (Liu et al., Proc Natl Acad Sci USA 96: 9563-9567,1999; Keith et al., J Cell Biol 139: 1281-1292, 1997). The cells wereharvested and lysed 20 hours after transfection, and aliquots wereloaded onto SDS/10-20% PAGE gels. The plasmid-encoded proteins werevisualized by immunoblotting with anti-Bcl-x_(L) mAb (Trevigen,Gaithersburg, Md.) and developed by using enhanced chemiluminescence(Amersham Pharmacia).

C. Protein Expression, Purification, SDS-Page and Western Blotting

The proteins LF_(n), LF_(n)-Bcl-x_(L) and Bcl-x_(L) from codons 1 to 209were individually expressed in E. coli BL21 (DE3) (Novagen, Inc.) andpurified with a His-Tag binding purification kit (Novagen, Inc.). Thetransformed BL21(DE3) was cultured at 37° C. in LB medium until theOD600 reached 0.5-0.8, and treated with 1 mM IPTG, and then cultured for3 more hours. The cells was pelleted, suspended in 1×His-Tag bindingbuffer with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM aprotininand 1 mM leupeptin, and disrupted with French Press. The cytosol wasseparated from cell debris and undisrupted cells by centrifugation at20,000×g for 30 minutes and loaded on the His•Tag binding column. Theeluted proteins were dialyzed against 1×PBS and sterilized with 0.22-umfilter. Protective antigen (PA) was purified as reported (Milne et al.,Mol. Microbiol. 15: 661-666, 1995). The proteins were run on SDS-PAGEgels, and stained with Coomassie Blue or visualized by immunoblottingwith anti-Bcl-x_(L) antibody, and developed as above.

D. J744 Macrophage-Like Cell Culture, Treatment and Apoptosis Assay

J744 macrophage-like cells at 10⁵/ml were placed in 96-well plates (100μl per well), and cultured overnight in RPMI 1640 with 10% FCS. Thecells were treated with PBS, 0.1 μM staurosporine alone or 0.1 μMstaurosporine along with the different combinations of the proteinsLF_(n)-Bcl-x_(L) (28 μg/ml), PA (33 μg/ml), LF_(n) (28 μg/ml) andBcl-x_(L) (28 μg/ml). The apoptotic and living cells were counted withHoechst dye no. 33342 as reported (Liu et al., Proc Natl Acad Sci USA96: 9563-9567, 1999).

E. Optic Nerve Section and Intra-Ocular Protein Injection

The P0 pups of Fisher 344 rat strain were used for the present study. P0is defined as the day of birth. The intracranial lesion of unilateraloptic nerve was performed as reported (Rabachi et al., J Neurosci. 14:5292-301, 1994). Briefly, a P0 pup was anesthetized by hypothermia.Under a dissecting microscope, an incision over the right eye was cutand a piece of bone flipped up. The right optic nerve was sectionedafter suctioning the overlying cerebral cortex. The section site ofoptic nerve is about 3 mm away from the eyeball. A piece of gelfoam wasput in the hole, and the flipped bone replaced, and the incisionrepaired with SUPERGLUE™. Immediately after the operation, seven, tenand four mice were respectively treated with administration of PBS,LF_(n)-Bcl-x_(L) (0.65 μg) plus PA (0.35 μg) and PA (0.35 μg) in avolume of 350 nanoliters (nl) per eye through ora serrata into theposterior chamber of the right eyes by using a micro-injector with apulled micropipette. The pups were warmed up with a light lamp until therecovery, and then sent back to the mother. Four pups from the samelitters, which were not operated and not treated, were used for normalcontrol.

F. Histology

About 24 hours after sectioning of the optic nerve, the right eyes wereremoved under deep anesthesia with sodium pentobarbital, fixed in 4%paraformaldehyde for approximately 30 hours, embedded in paraffin andcut at 6 μm. The eyes taken from the normal pups in the same litterswere processed in the same way to serve as controls. The sections wererehydrated, stained with 0.2% cresyl violet, dehydrated, and mountedwith DPX mountant. The number of pyknotic cells and the number of livingcells were counted by the use of 40× objective in the entire retinalganglion cell layer of three sections per retina. The pyknotic cellswere identified as reported (Rabachi et al., J. Neurosci. 14: 5292-301,1994). The values were presented as the percentage of pyknotic cellsversus total cells per retina (FIG. 12).

G. Results

The PA protein from the Anthrax bacillus binds cell receptors and canmediate the delivery of the anthrax LF protein to the cell cytosol whereLF effects toxicity to cells. The N-terminal domain of LF binds to PA.When exogenous peptides are fused to the N-terminal domain of LF(LF_(n)), they can be delivered to the cell cytosol by PA. Deletion ofthe C-terminal region of LF prevents toxicity to cells. To deliverBcl-x_(L) to cells, the N-terminal 255 amino acids of LF were fused toBcl-x_(L) without including the C-terminal 24 hydrophobic amino acids ofBcl-x_(L), as shown schematically in FIG. 8. The nucleotide and aminoacid sequences of the fusion protein, LF_(n)-Bcl-x_(L), are shown in SEQID NOs: 7 and 8. The fusion protein was expressed in E. coli andpurified to near homogeneity.

The bioactivity of the LF_(n)-Bcl-x_(L) was explored in J774 cells intissue culture. LF_(n)-Bcl-x_(L), at 28 micrograms per ml plus PA at 33micrograms per ml was added to the media of cells at the time ofapoptosis induction with 0.1 μM staurosporine (STS). Cells treated withstaurosporine alone died by apoptosis over the following 36 hours asshown in FIG. 9. When the cells were treated with LF_(n)-Bcl-x_(L) plusPA, most of the cell death was inhibited.

Controls were performed to explore the requirements for apoptosisinhibition. FIG. 10 shows data demonstrating that J774 cells treatedwith LF_(n) alone, Bcl-x_(L) alone, LF_(n)-Bcl-x_(L) without PA, and PAwithout LF_(n)-Bcl-x_(L) were not protected from apoptosis induced bystaurosporine, whereas LF_(n)-Bcl-x_(L) plus PA prevented more than halfof the cell death. Jurkat cells were also protected from apoptosis byLF_(n)-Bcl-x_(L) plus PA (FIG. 11).

This new strategy to block cell death was explored in an in vivo modelof neuron apoptosis. Retinal ganglion cells were axotomized andimmediately afterwards a mixture containing 0.35 μg of PA and 0.65 μg ofLF_(n)-Bcl-x_(L) was injected into the eye. Control mice were either notaxotomized, axotomized and injected with PBS, or axotomized and injectedwith PA alone. Mice were sacrificed 24 hours later, and the eyesexamined histologically. An increase in pyknotic cells, i.e., apoptoticcells (Rabachi et al., J Neurosci. 14: 5292-301, 1994), occurs in theganglion layer 24 hours after axotomy. However, when eyes are injectedwith LF_(n)-Bcl-x_(L) and PA, much of the cell death is inhibited. PAalone did not prevent cell death. To quantitate the extent of celldeath, the number of living and pyknotic cells in three entire ganglionlayers in one eye from each of 4-10 mice was counted. The quantifiedresults are shown in FIG. 12. LF_(n)-Bcl-x_(L) inhibited more than halfof the cell death due to neuron axotomy in vivo.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiments are only preferred examples of the invention, and should notbe taken as limitations on its scope. Rather, the scope of the inventionis defined by the following claims. We therefore claim as our inventionall that comes within the scope and spirit of these claims.

TABLE 2 Inhibition of Apoptosis by Bcl-x_(L)-DTR Concentration of Timeof STS Apoptosis Apoptosis Bcl-x_(L)-DTR Treatment Prevention DT Cellline inducer (μM) (Hrs) (%*) IO₅₀ (M) Cos-7 0.1 μM STS 4.8 12 58.410⁻¹²-10⁻¹¹ (monkey kidney) U251 0.1 μM STS 4.68 16 57.5 10⁻¹²-10⁻¹¹(human glioma) HeLa 0.2 μM STS 2.17 10 32.4 10⁻¹²-10⁻¹¹ (human cervicalCa) Jurkat 0.1 μM STS 4.68 12 21.2  10⁻⁹ (human T leukemia) 9L 0.1 μMSTS 4.68 12 −5.4 >10⁻⁷ (rat gliosarcoma) WEH7.1 0.1 μM STS 4.68 12 0.5>10⁻⁷ (mouse T lymphoma) *Apoptotic cells were counted with Hoechst dyeno. 33342 and the percent prevention from apoptosis was calculated as 1− (number of apoptotic cells with STS and Bcl-x_(L)-DTR − number ofapoptotic cells without STS and Bcl-x_(L)-DTR)/(number of apoptoticcells with STS − number of apoptotic cells without STS andBcl-x_(L)-DTR) except for the non-adherent Jurkat and WEHI7.1 cellswhich were counted by trypan blue dye exclusion and % apoptosisprevention calculated as (number of living cells with STS andBcl-x_(L)-DTR − number of living cells with STS)/(number of living cellswithout STS and Bcl-x_(L)-DTR).

TABLE 3 Brefeldin A prevents Bcl-x_(L)-DTR blockade of apoptosis 0.1 μMSTS + PBS 0.1 μM STS 2.24 μM Bcl-x_(L)-DTR Bcl-x_(L)-DTR Cell 1 24 1156% protection death (%) 2 μM 0.1 μM 0.1 μM STS + brefeldin STS +2 μM 2μM brefeldin A + Bcl-x_(L)-DTR + A brefeldin A 2.24 μM Bcl-x_(L)-DTRbrefeldin A Cell 2 35 32 9% protection death (%) Apoptotic cells werecounted with Hoechst dye no. 33342 14 hours after addition of STS and/orbrefeldin A minutes after Bcl-x_(L)-DTR was added to Cos-7 cells. Theprotection percentage was calculated as 1 − (number of apoptotic cellswith STS and Bcl-x_(L)-DTR − number of apoptotic cells without STS andBcl-x_(L)-DTR)/(number of apoptotic cells with STS − number of apoptoticcells without STS and Bcl-x_(L)-DTR).

1. A method for inhibiting apoptosis in a target cell, comprisingcontacting the target cell with an amount of a functionalapoptosis-inhibiting fusion protein sufficient to inhibit apoptosis inthe target cell, wherein the fusion protein comprises: a first domaincapable of inhibiting apoptosis in the target cell; and a second domaincapable of specifically targeting the fusion protein to the target cell,and wherein the fusion protein integrates into or otherwise crosses acellular membrane of the target cell upon binding.
 2. The method ofclaim 1, wherein the protein is administered in the form of apharmaceutical composition.
 3. The method of claim 1, wherein the targetcell is a neuron.
 4. The protein of claim 3, wherein the neuron is aretinal ganglion cell.
 5. The method of claim 1, wherein the firstdomain of the fusion protein is a Bcl-2 family protein, or a variant orfragment thereof that inhibits apoptosis in the target cell to which theprotein is exposed, and wherein the amount is sufficient to inhibitapoptosis.
 6. The method of claim 5, wherein the first domain of thefusion protein is Bcl-x_(L), or a variant or fragment thereof thatinhibits apoptosis in the target cell to which the protein is exposed,and wherein the amount is sufficient to inhibit apoptosis.
 7. The methodof claim 1, wherein the fusion protein comprises an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence shownin SEQ ID NO: 2 (Bcl-x_(L)-DTR); (b) the amino acid sequence shown inSEQ ID NO: 8 (LF_(n)-Bcl-x_(L)); and (c) amino acid sequences thatdiffer from those specified in (a) or (b) by one or more conservativeamino acid substitutions, but which retain targeting andapoptosis-inhibiting abilities.
 8. The method of claim 1, wherein thetarget cell is in vivo in a subject.
 9. The method of claim 1, whereinthe target cell is in vivo in a subject and the fusion protein isadministered to the subject after transient ischemic neuronal injury.10. The method of claim 9, wherein the transient ischemic neuronalinjury comprises a spinal cord injury.
 11. The method of claim 9,wherein the protein is administered in the form of a pharmaceuticalcomposition.
 12. The method of claim 9, further comprising the step ofco-administering an agent selected from the group consisting of achemotherapeutic agent, an anti-inflammatory agent, an anti-viral agent,and an antibiotic agent.
 13. The method of claim 1, wherein the seconddomain comprises a receptor-binding domain derived from a bacterialtoxin, a monoclonal antibody, a growth factor, or a cytokine.
 14. Themethod of claim 13, wherein the second binding domain comprises areceptor-binding domain derived from diphtheria toxin or anthrax toxin.15. The method of claim 13, wherein the second binding domain comprisesa receptor-binding domain derived from epidermal growth factor
 16. Themethod of claim 13, wherein the receptor-binding domain comprisesdiphtheria toxin receptor binding domain, or a variant or fragmentthereof that targets the fusion protein to the target cell to which theprotein is exposed.
 17. The method of claim 14, wherein the seconddomain further comprises a translocation domain of diphtheria toxin.